抗菌肽Cec4a的重组表达和抗菌活性研究*

目的:构建Cec4a的原核重组表达体系,通过诱导表达、酶切纯化获得重组蛋白,并检测产物的抗菌活性。方法:基于Cec4a的序列设计引物,克隆Cec4a基因的DNA片段。利用原核表达载体(pCold-SUMO)构建重组原核表达质粒,并将其转化到大肠杆菌C41(DE3)等感受态细胞,使用IPTG进行诱导表达。通过Ni-NTA亲和层析柱纯化,获得含有His-SUMO标签的重组Cec4a融合蛋白。在SUMO蛋白酶酶切后,再次使用Ni-NTA亲和层析纯化,得到目的蛋白,最后用鲍曼不动杆菌(ATCC19606)作为指示菌检测表达产物的抗菌活性。结果:成功构建pCold-SUMO-Cec4a原核表达质粒,测序分析其序列与预期结果一致。Cec4a融合蛋白表达量为42.8mg/L,纯化后的Cec4a重组蛋白对鲍曼不动杆菌的MIC为4 μg/mL。结论:通过原核表达,并经Ni-NTA亲和层析纯化,获得了具有抗菌活性的重组蛋白Cec4a,为研究Cec4a的生物活性、抗菌机制及应用奠定了基础。

Recombinant Expression and Detection of Antimicrobial Activity of Cec4a

Objective: To construct the recombinant expression system of Cec4a, and to obtain the recombinant protein by induced expression and detect the antibacterial activity of the product. Methods: Based on the primers designed according to the sequence of Cec4a, the mature peptide part of Cec4a gene was amplified by PCR. Recombinant prokaryotic expression plasmid was constructed using prokaryotic expression vector (pCold-SUMO) and transformed into E. coli C41 (DE3) competent cells, which were induced by IPTG. His-SUMO labeled recombinant Cec4a fusion protein was purified by Ni-NTA affinity chromatography. The target protein was purified by Ni-NTA affinity chromatography after SUMO protease digestion. Acinetobacter baumannii (ATCC19606) was used to detect the antibacterial activity of the product. Results: pCold-SUMO-Cec4a prokaryotic expression plasmid was successfully constructed, and the sequencing analysis was consistent with the expected results. The expression level of Cec4a fusion protein was 42.8mg/L, and the MIC of purified Cec4a recombinant protein against Acinetobacter baumannii was 4 μg/mL. Conclusion: The recombinant Cec4a protein with antibacterial activity was successfully constructed and purified by Ni-NTA affinity chromatography. It lays a foundation for further study on the biological activity, the relationship between the structure and function of Cec4a.